Myocardial tissue is enriched with myoglobin, which exists predominantly as oxymyoglobin under aerobic conditions. During ischemia, deoxymyoglobin accumulates due to decreased oxygen concentration. Nitric oxide has a very high affinity for deoxymyoglobin and forms nitrosylmyoglobin (MbNO), which can be detected by electron spin resonance (ESR). Previously, it has been shown nitric oxide donor S-nitrosoglutathione (GSNO) improves the postischemic functional recovery in crystalloid buffer-perfused isolated rat hearts subjected to cardioplegic ischemia. Supplementation of cardioplegic solution with nitronyl nitroxide, a scavenger of nitric oxide, antagonized this protective effect. Using low temperature ESR, we have detected MbNO in rat hearts subjected to cardioplegic ischemia in the presence of GSNO (20-200 fmol/l). Nitronyl nitroxide (400 fmol/l) prevented the formation of MbNO induced by 20 fmol/l GSNO during ischemia. During aerobic reperfusion, MbNO signal intensity gradually decreased, but persisted for up to 30 min or aerobic reperfusion. We conclude that MbNO is a sensitive intracellular marker of nitric oxide release in myocardial tissues. Implications of MbNO formation are discussed with respect to cardioprotection during ischemia- and reperfusion-induced myocardial injury.